Major histocompatibility complex class I (MHC-I) molecules are central to the immune response against virus-infected and neoplastic cells, the rejection of foreign tissue grafts, and the adverse reaction in a number of autoimmune diseases. MHC-I molecules function as scavenger receptors that bind a variety of peptides from degraded intracellular proteins and present these to the antigen-specific receptor on T lymphocytes. The intracellular pathways that result in this MHC-I/peptide complex are not well understood. Preliminary studies of transfected thymoma cells indicate that glycosyl phosphatidylinositol (GPI)-anchored MHC-I molecules are defective in antigen processing and/or T cell recognition. This defect corresponds to differences in intracellular trafficking and membrane topology between GPI- anchored and transmembrane MHC-I. LLC-PK1 pig kidney cells were therefore transfected with the same constructs to understand the pathways these MHC-I isoforms follow in polarized epithelia, which express endogenous transmembrane MHC-I molecules on their basolateral surface and sort GPI- anchored proteins to their apical surface. Preliminary results indicate that the sorting signals for MHC-I are comprised of sequences in the anchor/cytoplasmic region as well as the extracellular domains. Therefore, this is a powerful system with which to investigate the relationship between intracellular trafficking and antigen presentation. This project aims to compare the polarized expression of GPI-anchored, transmembrane, and secreted forms of MHC-I molecules in LLC-PK1 cells doubly transfected with a panel of MHC-I constructs, using confocal laser scanning and electron microscopy, and biochemical methods involving membrane-selective biotinylation of metabolically labeled cells. Thus, it will be possible to study the exocytic and endocytic pathways of molecules differing in both extracellular domains and membrane anchors, and the sorting signals responsible for their polarized expression, in the same cell. In addition, preliminary studies demonstrate differences in the intracellular distribution of GPI-anchored and transmembrane MHC-I. Therefore, the endocytic and exocytic compartments containing these proteins will be characterized to determine where the intracellular pathways of GPI-anchored, transmembrane, and secreted MHC-I molecules converge and diverge. These pathways will be further studied by specifically perturbing or blocking intracellular trafficking with low temperature, or with drugs that modulate intracellular pH, microtubule stability, or lipid biosynthesis and transport. Accomplishment of these goals should elucidate pathways for generating and maintaining epithelial cell polarity, and increase our understanding of MHC-I trafficking. Both of these processes are altered in a number of disease states such as carcinoma and autoimmune nephritis.